Tumor hypoxia can compromise the effectiveness of radiation treatment and chemotherapy by proliferation dependent cytotoxins. On the other hand, hypoxic tumor cells are potential targets for selective chemotherapy by hypoxic cell cytotoxins. Rational intervention directed at hypoxic tumor cells by hyperbaric oxygen, carbogen breathing, hypoxic cell radiosensitizers, perfluorohydrocarbon emulsions or hypoxic cell cytotoxins will require a convenient way of measuring changes in hypoxia in individual tumors in particular patients. This revised competing continuation grant application proposes that the hypoxic cell marker, Pimonidazole, can serve the purpose of measuring tumor hypoxia in a clinical setting. The hypoxia cell marker approach depends on the fact that, at low oxygen concentrations (<10 mm Hg partial pressure), cellular redox enzymes efficiently activate nitroaromatic compounds in a way which leads to their irreversible binding to cellular macromolecules. The tissue bound nitroaromatic compounds become markers of tumor hypoxia with a resolution on the scale of single cells. When suitably labeled, the markers can be detected by autoradiography, scintillation counting, single photon emission tomography, positron emission transaxial tomography, magnetic resonance spectroscopy or immunochemistry methods. In the immunochemistry method, which is the focus of this grant application, hypoxic cells can be detected by means of fluorescent or colorimetric immunostaining of tumor sections (microscopy), enzyme linked immunosorbent assay (enzyme digested tissue biopsy) or flow cytometry (disaggregated tissue biopsy). The six Specific Aims of the grant application are designed to broaden the scientific basis of the hypoxia marker approach. Specifically, the synthesis of large quantities of Pimonidazole will be carried out as well as the syntheses of smaller amounts of other markers for mechanistic studies. The chemical nature of hypoxia marker binding to hypoxic cells will be investigated. The effect of intracellular pH on subcellular localization of Pimonidazole will be studied. The rate of catabolism of Pimonidazole adducts in hypoxic cells will be followed and compared with the rates of catabolism for other hypoxia markers. A full characterization of the oxygen dependence of Pimonidazole binding to hypoxic cells will be made. Monoclonal antibody reagents to Misonidazole will be prepared to complement those already available for Pimonidazole.